Plasmid

Part:BBa_K4876010

Designed by: Jin Luming   Group: iGEM23_Zhejiang-United   (2023-09-28)


p15A-cas9-op-λ-Red


iGEM Team Metropolis-Shanghai 2023

Composite Part BBa_K4876010 (p15A-cas9-op)

Engineering Principle

Nissle 1917 is an E. coli strain with probiotic properties, but for certain applications, it is necessary to knock out specific genes[1]. Since CRISPR-Cas systems have high target DNA specificity and programmability, they can serve as gene editing tools[2]. The development and optimization of such tools can facilitate the construction of engineered cell lines for diverse production needs while accelerating the exploration of biological systems. However, unwanted escapers that do not undergo the intended editing frequently arise during CRISPR-Cas-mediated microbial genome editing, reducing editing efficiency[3].

Therefore, this project aims to construct a highly efficient CRISPR-Cas9-based genome editing tool for E. coli Nissle 1917 by increasing Cas9 copy number and codon optimization, lowering escape rates to improve editing efficiency, and providing a reference for other genome editing tools. Herein, we constructed two Cas9 systems to test the gene editing efficiency in E. coli Nissle 1917, one of which was designed by Prof. LI QI[3], and the other was redesigned by codon optimization.

Construction Design

BBa _ K4876010 ( p15A-cas9-op ) is composed of BBa _ K4876003 ( cas9-op ), BBa _ K4876005 ( λ-Red ), and BBa _ K4876015 ( p15A ). The Cas9 gene used in this project is from Streptococcus. Due to species differences, some rare E. coli tRNAs may be heavily used in the original sequence, causing slow host growth. Codon optimization avoids rare tRNA usage so bacteria can grow faster while expressing the same product. We named the codon-optimized Cas9 as Cas9-op and constructed the p15A-Cas9-op-λ-Red plasmid (Figure 1).

Figure 1
Figure 1. Expression frame of the p15A-Cas9-op-λ-Red plasmid.

Experimental Approach

To construct p15A-Cas9-op-λ-Red, we amplified p15A and Cas9-op-λ-Red fragments using primers with homology arms and recombined them using a cloning kit. Results showed successful amplification and construction verified by colony PCR and sequencing (Figure 2).

Figure 2
Figure 2. The construction results of p15A-Cas9-op-λ-Red. (A) Transformation results. (B) Sequencing results.

Characterization/Measurement

Comparing to p15A-Cas9-λ-Red (BBa_K4876011), after Cas9 codon optimization, p15A-Cas9-op-λ-Red (BBa_K4876010) knocked out both gntT and lacZ at 100% efficiency which is higher than p15A-Cas9 (Figure 3).

Figure 3
Figure 3. Editing efficiency results of p15A-Cas9 and p15A-Cas9-op in E. coli Nissle 1917.

We further studied the editing efficiencies of p15A-Cas9-op-λ-Red for the gntT gene and lacZ gene by adjusting the length of the repair homology arms. The results are shown in Figure 4, for the gntT gene, the editing efficiencies of the repair homology arms of 100-200 bp were 0, while it reached 100% when the lengths were extended to 300-500 bp. Similarly, for the lacZ gene, the editing efficiency of the repair homology arm of 100 bp was 0, which increased to 80% for 200-300 bp, and reached 100% for 400-500 bp.

Figure 4
Figure 4. Editing efficiency results of Cas9-op with different lengths of homology arms in E. coli Nissle 1917.

References:

  1. Buddenborg C., Daudel D., Liebrecht S., et al. Development of a tripartite vector system for live oral immunization using a gram-negative probiotic carrier [J]. Int J Med Microbiol, 2008, 298(1-2): 105-114.
  2. Vento J.M., Crook N., Beisel C.L. Barriers to genome editing with crispr in bacteria [J]. J Ind Microbiol Biotechnol, 2019, 46(9-10): 1327-1341.
  3. Li Q., Sun M., Lv L., et al. Improving the editing efficiency of crispr-Cas9 by reducing the generation of escapers based on the surviving mechanism [J]. ACS Synthetic Biology, 2023, 12(3): 672-680.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 6863
    Illegal NheI site found at 9344
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3244
    Illegal BglII site found at 3703
    Illegal BglII site found at 5632
    Illegal BamHI site found at 5434
    Illegal BamHI site found at 11620
    Illegal BamHI site found at 12480
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 5705
    Illegal NgoMIV site found at 12506
    Illegal AgeI site found at 1397
    Illegal AgeI site found at 6306
    Illegal AgeI site found at 6949
    Illegal AgeI site found at 7273
    Illegal AgeI site found at 12829
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2225
    Illegal SapI.rc site found at 5125


[edit]
Categories
Parameters
None